Structure-based designed herbicide resistant products

ABSTRACT

Disclosed herein are structure-based modelling methods for the preparation of acetohydroxy acid synthase (AHAS) variants, including those that exhibit selectively increased resistance to herbicides such as imidazoline herbicides and AHAS inhibiting herbicides. The invention encompasses isolated DNAs encoding such variants, vectors that include the DNAs, and methods for producing the variant polypeptides and herbicide resistant plants containing specific AHAS gene mutations. Methods for weed control in crops are also provided.

This application is a continuation of U.S. Ser. No. 09/367,512 filedAug. 17, 2000 now U.S. Pat. No. 6,576,455, which is a continuation ofPCT/US96/05782 filed Apr. 19, 1996, and a divisional of U.S. Ser. No.08/455,355 filed on May 31, 1995, issued as U.S. Pat. No. 5,928,937,which is a continuation-in-part of Ser. No. 08/426,125 filed Apr. 20,1995, issued as U.S Pat. No. 5,853,973.

FIELD OF THE INVENTION

This invention pertains to structure-based modelling and design ofvariants of acetohydroxy acid synthase (AHAS) that are resistant toimidazolinones and other herbicides, the AHAS inhibiting herbicides,AHAS variants themselves, DNA encoding these variants, plants expressingthese variants, and methods of weed management.

BACKGROUND OF THE INVENTION

Acetohydroxy acid synthase (AHAS) is an enzyme that catalyzes theinitial step in the biosynthesis of isoleucine, leucine, and valine inbacteria, yeast, and plants. For example, the mature AHAS from Zea Maysis approximately a 599-amino acid protein that is localized in thechloroplast (see FIG. 1; SEQ ID NO:1). The enzyme utilizes thiaminepyrophosphate (TPP) and flavin adenine dinucleotide (FAD) as cofactorsand pyruvate as a substrate to foam acetolateate. The enzyme alsocatalyzes the condensation of pyruvate and 2-ketobutyrate to formacetohydroxybutyrate. AHAS is also known as acetolactate synthase oracetolactate pyruvate lyase (carboxylating), and is designated EC4.1.3.18. The active enzyme is probably at least a homodimers. Ibdah etal. (Protein Science, 3:479-S, 1994), in an abstract, disclose one modelfor the active site of AHAS.

A variety of herbicides including imidazolinone compounds such asimazethapyr (PURSUIT®—American Cyanamid Company-Wayne, N.J.),sulfonylurea-based compounds such as sulfometuron methyl (OUST®—E. I. duPont de Nemours and Company-Wilmington, Del.), triazolopyrimidinesulfonamides (Broadstrike™—Dow Elanco; see Gerwick, et al., Pestic. Sci.29:357-364, 1990), sulfamoylureas (Rodaway et al., Mechanisms ofSelectively of Ac 322,140 in Paddy Rice, Wheat and Barley, Proceedingsof the Brighton Crop Protection Conference-Weeds, 1993),pyrimidyl-oxy-benzoic acids (STABLE®—Kumiai Chemical Industry Company,E. I. du Pont de Nemours and Company; see, The Pesticide Manual 10th Ed.pp. 888-889, Clive Tomlin, Ed., British Crop Protection Council, 49Downing Street, Farmham, Surrey G49 7PH, UNITED KINGDOM), andsulfonylcarboxamides (Alvarado et al., U.S. Pat. No. 4,883,914) act byinhibiting AHAS enzymatic activity. (See, Chaleff et al., Science224:1443, 1984; LaRossa et al., J. Biol. Chem. 259:8753, 1984; Ray,Plant Physiol. 75:827, 1984; Shaner et al., Plant Physiol. 76:545,1984). These herbicides are highly effective and environmentally benign.Their use in agriculture, however, is limited by their lack ofselectivity, since crops as well as undesirable weeds are sensitive tothe phytotoxic effects of these herbicides.

Bedbrook et al., U.S. Pat. Nos. 5,013,659, 5,141,870, and 5,378,824,disclose several sulfonylurea resistant AHAS variants. However, thesevariants were either obtained by mutagenizing plants, seeds, or cellsand selecting for herbicide-resistant mutants, or were derived from suchmutants. This approach is unpredictable in that it relies (at leastinitially) on the random chance introduction of a relevant mutation,rather than a rational design approach based on a structural model ofthe target protein.

Thus, there is still a need in the art for methods and compositions thatprovide selective wide spectrum and/or specific herbicide resistance incultivated crops. The present inventors have discovered that selectiveherbicide resistant variant forms of AHAS and plants containing the samecan be prepared by structure-based modelling of AHAS against pyruvateoxidase (POX), identifying an herbicide binding pocket or pockets on theAHAS model, and designing specific mutations that alter the affinity ofthe herbicide for the binding pocket. These variants and plants are notinhibited or killed by one or more classes of herbicides and retainsufficient AHAS enzymatic activity to support crop growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a 600 amino acid sequence (SEQ ID NO:1)corresponding to the approximately 599 amino acid sequence ofacetohydroxy acid synthase (AHAS) from Zea Mays which is given as anexample of a plant AHAS enzyme. The sequence does not include a transitsequence, and the extra glycine is vestigial from a thrombin cleavagesite. Residues Met53, Arg128, and Phe135 are shown in bold.

FIG. 2 is an illustration of the alignment of the sequence of maize AHASand pyruvate oxidase (POX) from Lactobacillus planarum (SEQ ID NO:2).

FIG. 3 is a schematic representation of the secondary structure of anAHAS subunit. Regular secondary structure elements, α-helices andβ-sheets, are depicted as circles and ellipses, respectively, and arenumbered separately for each of the three domains within a subunit.Loops and coiled regions are represented by black lines, with numbersrepresenting the approximate beginnings and ends of the elements. Thelocations of cofactor binding sites and known mutation sites areindicated by octahedrons and stars, respectively.

FIG. 4 is an illustration of a computer-generated model of the activesite of maize AHAS with imazethapyr (PURSUIT® herbicide) modeled intothe binding pocket.

FIG. 5 is an illustration of the homology among AHAS amino acidsequences derived from different plant species. pAC 751 is maize als 2AHAS isozyme as expressed from the pAC 751 E. Coli expression vector asin FIG. 1 (SEQ ID NO:1); Maize als 2 is the maize als 2 AHAS isozyme(SEQ ID NO:3); Maize als 1 is the maize als 1 AHAS isozyme (SEQ IDNO:4); Tobac 1 is the tobacco AHAS SuRA isozyme (SEQ ID NO:5); Tobac 2is the tobacco AHAS SuRB isozyme (SEQ ID NO:6); Athesr 12 is theArabidopsis thaliana Csr 1.2 AHAS gene (SEQ ID NO:7); Bnaal 3 is theBrassica napus AHAS III isozyme (SEQ ID NO:8); and Bnaal 2 is theBrassica napus AHAS II isozyme (SEQ ID NO:9).

pAC 751 and Maize als 2 are identical genes except that Maize als 2starts at the beginning of the transit sequence and pAC 751 starts atthe putative mature N-terminal site with an additional glycine at theN-terminal due to the thrombin recognition sequence in the pGEX-2Texpression vector. The N-terminal glycine is not a natural amino acid atthat position.

Amino acid sequence alignments of the AHAS proteins were generated byPILEUP (GCG Package—Genetics Computer Group, Inc.,—University ResearchPark—Madison-Wis.). The consensus sequence was generated by PRETTY GCGPackage.

FIG. 6 is a photographic illustration of an SDS-polyacrylamide gelstained for protein showing purification of maize AHAS. The lanescontain (from left to right): A, Molecular weight markers; B, Crude E.coli cell extract; C, Glutathione-agarose affinity purified preparation;D, Thrombin digest of the affinity purified preparation; E, Second passthrough glutathione-agarose column and Sephacryl S-100 gel filtration.

FIG. 7 is a graphic illustration of the results of in vitro assays ofthe enzymatic activity of wild-type and mutant AHAS proteins in theabsence and in the presence of increasing concentrations of imazethapyr(PURSUIT® herbicide). The Y axis represents the % of activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 8 is a graphic illustration of the results of in vitro assays ofthe enzymatic activity of wild-type and mutant AHAS proteins in theabsence and presence of increasing concentrations of sulfometuron methyl(OUST® herbicide). The Y axis represents the % of activity of the mutantenzyme, wherein the 100% value is measured in the absence of inhibitor.

FIG. 9 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and the Met124Ile mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 10 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and the Met124His mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 11 is a graphic illustration of in vitro assays of the enzymaticactivity of wild-type Arabidopsis AHAS protein and Arg199Glu mutantArabidopsis AHAS protein in the absence and presence of increasingconcentrations of imazethapyr (PURSUIT® herbicide) and sulfometuronmethyl (OUST® herbicide). The Y axis represents the % activity of themutant enzyme, wherein the 100% value is measured in the absence ofinhibitor.

FIG. 12 is a schematic illustration of a DNA vector used for planttransformation, which contains the nptII gene (encoding kanamycinresistance) under the control of the 35S promoter and an AHAS gene (wildtype or variant) under the control of the Arabidopsis AHAS promoter.

FIG. 13 is a photograph showing the root development of tobacco plantstransformed with the Arabidopsis AHAS gene containing either theMet124Ile or Arg199Glu mutation and a non-transformed control. Plantswere grown for 18 days after transfer into medium containing 0.25 μMimazethapyr.

FIG. 14 is a photograph showing tobacco plants transformed with theArabidopsis AHAS gene containing either the Met124Ile, Met 124His, orArg199Glu mutation and a non-transformed control, which had been sprayedwith twice the field rate (100 g/ha) of imazethapyr.

FIG. 15 is a photograph showing the results of germination testsperformed in the presence of the herbicide CL 299,263 (imazamox), whichwere performed on seeds harvested from primary tobacco planttransformants that had been transformed with the Arabidopsis AHAS genecontaining either the Met124Ile, Met 124His, or Arg199Glu mutation.

SUMMARY OF THE INVENTION

The present invention provides a structure-based modelling method forthe production of herbicide resistant AHAS variant protein. The methodincludes:

-   -   (a) aligning a target AHAS protein on pyruvate oxidase template        or an AHAS modelling equivalent thereof to derive the        three-dimensional structure of the target AHAS protein;    -   (b) modelling one or more herbicides into the three-dimensional        structure to localize an herbicide binding pocket in the target        AHAS protein;    -   (c) selecting as a target for a mutation, at least one amino        acid position in the target AHAS protein, wherein the mutation        alters the affinity of at least one herbicide for the binding        pocket;    -   (d) mutating DNA encoding the target AHAS protein to produce a        mutated DNA encoding a variant AHAS containing the mutation,        such as, for example, at least one different amino acid, at the        position; and    -   (e) expressing the mutated DNA in a first cell, under conditions        in which the variant AHAS containing the mutation, such as, for        example, the different amino acid(s), at the position is        produced.

The method further may include:

-   -   (f) expressing DNA encoding wild-type AHAS protein parallel in a        second cell;    -   (g) purifying the wild-type and the variant AHAS proteins from        the cells;    -   (h) assaying the wild-type and the variant AHAS proteins for        catalytic activity in conversion of pyruvate to acetolactate or        in the condensation of pyruvate and 2-ketobutyrate to form        acetohydroxybutyrate, in the absence and in the presence of the        herbicide; and    -   (i) repeating steps (c)-(h), wherein the DNA encoding the AHAS        variant of step (e) is used as the AHAS-encoding DNA in step (c)        until a first herbicide resistant AHAS variant protein is        identified having:    -   (i) in the absence of the at least one herbicide,        -   (a) catalytic activity alone sufficient to maintain the            viability of a cell in which it is expressed; or        -   (b) catalytic activity in combination with any herbicide            resistant AHAS variant protein also expressed in the cell,            which may be the same as or different than the first AHAS            variant protein, sufficient to maintain the viability of a            cell in which it is expressed;        -   wherein the cell requires AHAS activity for viability; and        -   (ii) catalytic activity that is more resistant to the at            least one herbicide than is wild-type AHAS.

An alternate structure-based modelling method for the production ofherbicide resistant AHAS variant protein is also provided. This methodincludes:

-   -   (a) aligning a target AHAS protein on a first AHAS template        derived from a polypeptide having the sequence of FIG. 1 or a        functional equivalent thereof to derive the three-dimensional        structure of the target AHAS protein;    -   (b) modelling one or more herbicides into the three-dimensional        structure to localize an herbicide binding pocket in the target        AHAS protein;    -   (c) selecting as a target for a mutation, at least one amino        acid position in the target AHAS protein, wherein the mutation        alters the affinity of at least one herbicide for the binding        pocket;    -   (d) mutating DNA encoding the target AHAS protein to produce a        mutated DNA encoding a variant AHAS containing the mutation at        the position; and    -   (e) expressing the mutated DNA in a first cell, under conditions        in which the variant AHAS containing the mutation at the        position is produced.

This method can further include:

-   -   (f) expressing DNA encoding wild-type AHAS protein in parallel        in a second cell;    -   (g) purifying the wild-type and the variant AHAS protein from        the cells;    -   (h) assaying the wild-type and the variant AHAS protein for        catalytic activity in conversion of pyruvate to acetolactate or        in the condensation of pyruvate and 2-ketobutyrate to form        acetohydroxybutyrate, in the absence and in the presence of the        herbicide; and    -   (i) repeating steps (c)-(h), wherein the DNA encoding the AHAS        variant of step (e) is used as the AHAS-encoding DNA in step (c)        until a first herbicide resistant AHAS variant protein is        identified having:        -   (i) in the absence of the at least one herbicide,            -   (a) catalytic activity alone sufficient to maintain the                viability of a cell in which it is expressed; or            -   (b) catalytic activity in combination with any herbicide                resistant AHAS variant protein also expressed in the                cell, which may be the same as or different than the                first AHAS variant protein, sufficient to maintain the                viability of a cell in which it is expressed;            -   wherein the cell requires AHAS activity for viability;                and        -   (ii) catalytic activity that is more resistant to the at            least one herbicide than is wild-type AHAS.

In another alternate embodiment, the method includes:

-   -   (a) aligning a target AHAS protein on a first AHAS template        having an identified herbicide binding pocket and having the        sequence of FIG. 1 or a functional equivalent thereof to derive        the three-dimensional structure of the target AHAS protein;    -   (b) selecting as a target for a mutation, at least one amino        acid position in the target AHAS protein, wherein the mutation        alters the affinity of at least one herbicide for the binding        pocket;    -   (c) mutating DNA encoding the target AHAS protein to produce a        mutated DNA encoding a variant AHAS containing the mutation at        the position; and    -   (d) expressing the mutated DNA in a first cell, under conditions        in which the variant AHAS containing the mutation at the        position is produced.

This method can further include:

-   -   (e) expressing DNA encoding wild-type target AHAS protein in        parallel in a second cell;    -   (f) purifying the wild-type and the variant AHAS protein from        the cells;    -   (g) assaying the wild-type and the variant AHAS protein for        catalytic activity in conversion of pyruvate to acetolactate or        in the condensation of pyruvate and 2-ketobutyrate to form        acetohydroxybutyrate, in the absence and in the presence of the        herbicide; and    -   (h) repeating steps (b)-(g), wherein the DNA encoding the AHAS        variant of step (d) is used as the AHAS-encoding DNA in step (b)        until a first herbicide resistant AHAS variant protein is        identified having:        -   (i) in the absence of the at least one herbicide,            -   (a) catalytic activity alone sufficient to maintain the                viability of a cell in which it is expressed; or            -   (b) catalytic activity in combination with any herbicide                resistant AHAS variant protein also expressed in the                cell, which may be the same as or different than the                first AHAS variant protein, sufficient to maintain the                viability of a cell in which it is expressed;            -   wherein the cell requires AHAS activity for viability;                and        -   (ii) catalytic activity that is more resistant to the at            least one herbicide than is wild-type AHAS.

In preferred embodiments of the above methods, the catalytic activity inthe absence of the herbicide is at least about 5% and most preferably ismore than about 20% of the catalytic activity of the wild-type AHAS.Where the herbicide is an imidazolinone herbicide, the herbicideresistant AHAS variant protein preferably has:

-   -   (i) catalytic activity in the absence of the herbicide of more        than about 20% of the catalytic activity of the wild-type AHAS;    -   (ii) catalytic activity that is relatively more resistant to the        presence of imidazolinone herbicides compared to wild-type AHAS;        and    -   (iii) catalytic activity that is relatively more sensitive to        the presence of sulfonylurea herbicides compared to        imidazolinone herbicides.

The present invention further provides isolated DNA encodingacetohydroxy acid synthase (AHAS) variant proteins, the variant proteinscomprising an AHAS protein modified by;

(i) substitution of at least one different amino acid residue at anamino acid residue of that sequence of FIG. 1 (SEQ ID NO:1) selectedfrom the group consisting of P48, G49, S52, M53, E54, A84, A95, T96,S97, G98, P99, G100, A101, V125, R127, R128, M129, I130, G131, T132,D133, F135, Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277,L278, G279, H281, G282, T283, V284, G300, V301, R302, F303, D304, R306,V307, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317, A318,1K319, I320, E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415,Q416, H417, Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438,L439, G440, A441, M442, G443, D467, G468, S469, L471, N473, L477, M479,Q495, H496, L497, G498, M499, V501, Q502, Q504, D505, D506, Y508, K509,A510, N511, R512, A513, H514, T515, S524, H572, Q573, E574, H575, V576,L577, P578, M579, I580, P581, G583, G584, functional equivalents of anyof the foregoing, and any combination of any of the foregoing;

(ii) deletion of up to 5 amino acid residues preceding, or up to 5 aminoacid residues following at least one amino acid residue of the sequenceof FIG. 1 (SEQ ID NO:1) selected from the group consisting of P48, G49,S52, M53, E54, A84, A95, T96, S97, G98, P99, G100, A101, V125, R127,R128, M129, I130, G131, T132, D133, F135, Q136, D186, I187, T259, T260,L261, M262, G263, R276, M277, L278, G279, H281, G282, T283, V284, V300,V301, R302, F303, D304, R306, V307, T308, G309, K310, I311, E312, A313,F314, A315, S316, R317, A318, K319, I320, E329, I330, K332, N333, K334,Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422,L434, S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468,S469, L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501, Q502,Q504, D505, R506, Y508, K509, A510, N511, R512, A513, H514, T515, S524,H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581, G583, G584,functional equivalents of any of the foregoing, and any combination ofany of the foregoing;

(iii) deletion of at least one amino acid residue or a functionalequivalent thereof between Q124 and H150 of the sequence of FIG. 1 (SEQID NO:1);

(iv) addition of at least one amino acid residue or a functionalequivalent thereof between Q124 and H150 of the sequence of FIG. 1 (SEQID NO:1);

(v) deletion of at least one amino acid residue or a functionalequivalent thereof between G300 and D324 of the sequence of FIG. 1 (SEQID NO:1);

(vi) addition of at least one amino acid residue or a functionalequivalent thereof between G300 and D324 of the sequence of FIG. 1 (SEQID NO:1); or

(vii) any combination of any of the foregoing.

In this numbering system, residue #2 corresponds to the putative aminoterminus of the mature protein, i.e., after removal of a chloroplasttargeting peptide.

The above modifications are directed to altering the ability of anherbicide, and preferably an imidazolinone-based herbicide, to inhibitthe enzymatic activity of the protein. In a preferred embodiment, theisolated DNA encodes an herbicide-resistant variant of AHAS. Alsoprovided are DNA vectors comprising DNA encoding these AHAS variants,variant AHAS proteins themselves, and cells, grown either in vivo or incell culture, that express the AHAS variants or comprise these vectors.

In another aspect, the present invention provides a method forconferring herbicide resistance on a cell or cells and particularly aplant cell or cells such as, for example, a seed. An AHAS gene,preferably the Arabidopsis thaliana AHAS gene, is mutated to alter theability of an herbicide to inhibit the enzymatic activity of the AHAS.The mutant gene is cloned into a compatible expression vector, and thegene is transformed into an herbicide-sensitive cell under conditions inwhich it is expressed at sufficient levels to confer herbicideresistance on the cell.

Also contemplated are methods for weed control, wherein a cropcontaining an herbicide resistant AHAS gene according to the presentinvention is cultivated and treated with a weed-controlling effectiveamount of the herbicide.

Also disclosed is a structure-based modelling method for the preparationof a first herbicide which inhibits AHAS activity. The method comprises:

-   -   (a) aligning a target AHAS protein on pyruvate oxidase template        or an AHAS modelling functional equivalent thereof to derive the        three-dimensional structure of the target AHAS protein;    -   (b) modelling a second herbicide having AHAS inhibiting activity        into the three-dimensional structure to derive the location,        structure, or a combination thereof of an herbicide binding        pocket in the target AHAS protein; and    -   (c) designing a non-peptidic first herbicide which will interact        with, and preferably will bind to, an AHAS activity inhibiting        effective portion of the binding pocket, wherein the first        herbicide inhibits the AHAS activity sufficiently to destroy the        viability of a cell which requires AHAS activity for viability.

An alternative structure-based modelling method for the production of afirst herbicide which inhibits AHAS activity, is also enclosed. Themethod comprises:

-   -   (a) aligning a target AHAS protein on a first AHAS template        derived from a polypeptide having the sequence of FIG. 1 or a        functional equivalent thereof, to derive the three-dimensional        structure of the target AHAS protein;    -   (b) modelling a second herbicide having AHAS inhibiting activity        into the three-dimensional structure to derive the location,        structure, or a combination thereof of an herbicide binding        pocket in the target AHAS protein; and    -   (c) designing a non-peptidic first herbicide which will interact        with, and preferably will bind to, an AHAS activity inhibiting        effective portion of the binding pocket, wherein the first        herbicide inhibits the AHAS activity sufficiently to destroy the        viability of a cell which requires AHAS activity for viability.

Preferably in each method, the first herbicide contains at least onefunctional group that interacts with a functional group of the bindingpocket.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the rational design or structure-basedmolecular modelling of modified versions of the enzyme AHAS and AHASinhibiting herbicides. These modified enzymes (AHAS variant proteins)are resistant to the action of herbicides. The present invention alsoencompasses DNAs that encode these variants, vectors that include theseDNAs, the AHAS variant proteins, and cells that express these variants.Additionally provided are methods for producing herbicide resistance inplants by expressing these variants and methods of weed control. The DNAand the AHAS variants of the present invention were discovered instudies that were based on molecular modelling of the structure of AHAS.

Rational Structure-Based Design of AHAS Variants and AHAS InhibitingHerbicides

Herbicide-resistant variants of AHAS according to the present inventionare useful in conferring herbicide resistance in plants and can bedesigned with the POX model, AHAS model, or functional equivalentsthereof, such as, for example, tranaketolases, carboligases, pyruvatedecarboxylase, proteins that bind FAD and/or TPP as a cofactor, or anyproteins which have structural features similar to POX and/or AHAS; withan AHAS model such as a model having the sequence of FIG. 1 (SEQ IDNO:1); or with a functional equivalent of the sequence of FIG. 1 (SEQ IDNO:1) including a variant modeled from a previous model. Proteins thatcan be used include any proteins having less than a root mean squaredeviation of less than 3.5 angstroms in their Cα carbons relative to anyof the above-listed molecules. AHAS directed herbicides can be similarlymodelled from these templates. A functional equivalent of an AHAS aminoavid sequence is a sequence having substantial, i.e., 60-70%, homology,particularly in conserved regions such as, for example, a putativebinding pocket, The degree of homology can be determined by simplealignment based on programs known in the art, such as, for example, GAPand PILEUP by GCG. Homology means identical amino acids or conservativesubstitutions. A functional equivalent of a particular amino acidresidue in the AHAS protein of FIG. 1 (SEQ ID NO: 1) is an amino acidresidue of another AHAS protein which when aligned with the sequence ofFIG. 1 (SEQ ID NO:1) by programs known in the art, such as, for example,GAP and PILEUP by GCG, is in the seine position an the amino acidresidue of FIG. 1 (SEQ ID NO:1).

Rational design steps typically include: (1) alignment of a target AHASprotein with a POX backbone or structure or an AHAS backbone orstructure; (2) optionally, and if the AHAS backbone has an identifiedherbicide binding pocket, modelling one or more herbicides into thethree-dimensional structure to localize an herbicide binding pocket inthe target protein; (3) selection of a mutation based upon the model;(4) site-directed mutagenesis; and (5) expression and purification ofthe variants. Additional steps can include (6) assaying of enzymaticproperties and (7) evaluation of suitable variants by comparison to theproperties of the wild-type AHAS. Each step is discussed separatelybelow.

1. Molecular Modelling

Molecular modelling (and particularly protein homology modelling)techniques can provide an understanding of the structure and activity ofa given protein. The structural model of a protein can be determineddirectly from experimental data such as x-ray crystallography,indirectly by homology modelling or the like, or combinations thereof(See White, et al., Annu. Rev. Biophys. Biomol. Struct., 23:349, 1994).Elucidation of the three-dimensional structure of AHAS provides a basisfor the development of a rational scheme for mutation of particularamino acid residues within AHAS that confer herbicide resistance on thepolypeptide.

Molecular modelling of the structure of Zea mays AHAS, using as atemplate the known X-ray crystal structure of related pyruvate oxidase(POX) from Lactobacillus plantarum, provides a three-dimensional modelof AHAS structure that is useful for the design of herbicide-resistantAHAS variants or AHAS inhibiting herbicides. This modelling proceduretakes advantage of the fact that AHAS and POX share a number ofbiochemical characteristics and may be derived from a common ancestralgene (Chang et al., J. Bacteriol. 170:3937, 1988).

Because of the high degree of cross-species homology in AHAS themodelled AHAS described herein or functional equivalents thereof canalso be used as templates for AHAS variant protein design.

Derivation of one model using interactive molecular graphics andalignments is described in detail below. The three-dimensional AHASstructure that results from this procedure predicts the approximatespatial organization of the active site of the enzyme and of the bindingsite or pocket of inhibitors such as herbicides including, but notlimited to, imidazolinone herbicides. The model is then refined andre-interpreted based on biochemical studies which are also describedbelow.

Protein homology modelling requires the alignment of the primarysequence of the protein under study with a second protein whose crystalstructure is known. Pyruvate oxidase (POX) was chosen for AHAS homologymodelling because POX and AHAS share a number of biochemicalcharacteristics. For example, both AHAS and POX share aspects ofenzymatic reaction mechanisms, as well as cofactor and metalrequirements. In both enzymes thiamine pyrophosphate (TPP), flavinadenine dinucleotide (FAD), and a divalent cation are required forenzymatic activity. FAD mediates a redox reaction during catalysis inPOX but presumably has only a structural function in AHAS, which ispossibly a vestigial remnant from the evolution of AHAS from POX. Bothenzymes utilize pyruvate as a substrate and form hydroxyethyl thiaminepyrophosphate as a stable reaction intermediate (Schloss, J. V. et al.In Biosynthesis of branched chain amino acids, Barak, Z. J. M., Chipman,D. M., Schloss, J. V. (eds) VCH Publishers, Weinheim, Germany, 1990).

Additionally, AHAS activity is present in chimeric POX-AHAS proteinsconsisting of the N-terminal half of POX and the C-terminal half ofAHAS, and there is a small degree of AHAS activity exhibited by POXitself. AHAS and POX also exhibit similar properties in solution (Risse,B. et al, Protein Sci. 1: 1699 and 1710, 1992; Singh, B. K., & Schmitt,G. K. (1989), FEBS Letters, 258: 113; Singh, B. K. et al. (1989) In:Prospects for Amino Acid Biosynthesis Inhibitors in Crop Protection andPharmaceutical Chemistry, (Lopping, L. G., et al., eds., BCPC Monographp. 87). With increasing protein concentration, both POX and AHAS undergostepwise transitions from monomers to dimers and tetramers. Increases inFAD concentration also induce higher orders of subunit assembly. Thetetrameric form of both proteins is most stable to heat and chemicaldenaturation.

Furthermore, the crystal structure of POX from Lactobacillus planarumhad been solved by Muller et al., Science 259:965, 1993. The presentinventors found that based in part upon the degree of physical,biochemical, and genetic homology between AHAS and POX, the X-raycrystal structure of POX could be used as a structural starting pointfor homology modelling of the AHAS structure.

AHAS and L. plantarum POX sequences were not similar enough for acompletely computerized alignment, however. Overall, only about 20% ofthe amino acids are identical, while about 50% of the residues are ofsimilar class (i.e. acidic, basic, aromatic, and the like). However, ifthe sequences are compared with respect to hydrophilic and hydrophobicresidue classifications, over 500 of the 600 amino acids match.Secondary structure predictions for AHAS (Holley et al., Proc. Natl.Acad. Sci. USA 86:152, 1989) revealed a strong similarity to the actualsecondary structure of POX. For nearly 70% of the residues, thepredicted AHAS secondary structure matches that of POX.

POX monomers consist of three domains, all having a central, parallelβ-sheet with crossovers consisting of α-helices and long loops. (Mulleret al., Science 259:965, 1993). The topology of the sheets differsbetween the domains, i.e. in the first and third domains, the strandsare assembled to the β-sheet in the sequence 2-1-3-4-6-5, while in theβ-sheet of the second domain, the sequence reads 3-2-1-4-5-6.

Computer generated alignments were based on secondary structureprediction and sequence homology. The conventional pair-wise sequencealignment method described by Needleman and Wunch, J. Mol. Biol, 48:443, 1970, was used. Two sequences were aligned to maximize thealignment score. The alignment score (homology score) is the sum of thescores for all pairs of aligned residues, plus an optional penalty forthe introduction of gaps into the alignment. The score for the alignmentof a pair of residues is a tabulated integer value. The homology scoringsystem is based on observing the frequency of divergence between a givenpair of residues. (M O Dayhoff, R M Schwartz & B C Orcutt “Atlas ofProtein Sequence and Structure” vol. 5 suppl. 3 pp. 345-362, 1978).

The alignments were further refined by repositioning gaps so as toconserve continuous regular secondary structures. Amino acidsubstitutions generated by evaluation of likely alignment schemes werecompared by means of interactive molecular graphics. Alignments with themost conservative substitutions with respect to the particularfunctionality of the amino acids within a given site were chosen. Thefinal alignment of both POX and AHAS is displayed in FIG. 2. Conservedclusters of residues were identified, in particular for the TPP bindingsite and for parts of the FAD binding site. The alignment revealed ahigh similarity between AHAS and POX for the first domain, for mostparts of the second domain, and for about half of the third domain. Mostof the regions that aligned poorly and may fold differently in POX andin AHAS were expected to be at the surface of the protein and were notinvolved in cofactor or inhibitor binding. The prediction of mutationsites is not substantially affected by small shifts in the alignment.

Most TPP binding residues are highly conserved between POX and AHAS(e.g. P48-G49-G50). In some cases, residues that were close to TPPdiffer between POX and AHAS but remain within a region that is highlyconserved (for example, residues 90-110). On the other hand, the FADbinding site appeared to be less conserved. Although some FAD bindingresidues were strongly conserved (for example, D325-I326-D327-P328),others clearly differed between AHAS and POX (for example, residues inthe loop from positions 278 to 285 are not homologous. A detailedanalysis revealed that, at least for some of the less-conserved contactsites, the interactions were mediated by the polypeptide backbone ratherthan by the side chains. Hence, conservation was only required for thepolypeptide fold and was not required for the amino acid sequence (forexample, the backbone of residues 258-263 binds the ribitol chain ofFAD). One half of the adenine and the isoalloxazine binding sitesclearly differ.

After aligning the primary structure, a homology model was built bytransposition of AHAS amino acid sequences to the POX templatestructure. Missing coordinates were built. stepwise using templates ofamino acid residues to complete undefined side chains. Data banksearches and energy minimization of small parts of the molecule wereused to complete the conformations of undefined loop regions. Thecofactors TPP and FAD were modeled into their binding pockets. Thismodel was then subjected to a complete, 5000 cycle energy minimization.All computer modelling was performed in an IRIS Indigo Elan R4000Workstation from Silicon Graphics Co. Interactive molecular modellingand energy-minimization were performed using Quanta/CHARMm 4.0 fromMolecular Simulations Inc. During this step, the conformation wasstable, indicating that no strongly disfavored interactions, such as,for example, close van der Waals contacts, had occurred. The results areshown schematically in FIG. 3.

Characteristics of Predicted AHAS Structure

Inspection of the modelled AHAS structure described above revealed thatmost of the protein folds with a backbone that is energeticallyreasonable, with most hydrophilic side chains accessible to the solvent.The surface of the β-sheets are smooth and accommodate the cross-overregions that are attached to them.

A model for dimeric AHAS was generated by duplicating the coordinates ofthe energy minimized monomeric AHAS and superimposing the two copies ontwo POX subunits using pairs of Cα coordinates as defined in thealignment scheme. The polypeptide chain of AHAS folds into threesimilarly folded domains composed of a six-stranded parallel β-sheetcore surrounded by long “loops” and α-helices. Two subunits areassembled such that the first domain of one subunit is in closeproximity to the cofactor-binding domains 2 and 3 of the other subunit.A solvent-filled space remains between the subunits at this site. Thispocket, which is defined by the confluence of the three domains, is theproposed entry site for the substrate. It is also proposed to be thebinding site for herbicides.

The inner surface of the binding pocket is outlined by the cofactors.The thiazol of TPP is positioned at the bottom of the pocket. Domain 3contributes to the inner surface of the pocket with a short α-helix thatpoints its axis towards the pyrophosphate of TPP, compensating thephosphate charges with its dipolar moment. This critical helix, whichstarts with G498, a “turn” residue in close contact with TPP, and whichends at F507, contains three known mutation sites for sulfonylurearesistance: V500, W503, and F507 (See, U.S. Pat. Nos. 5,013,659;5,141,870; and 5,378,824). In domain 1, the loop defined as P48-S52(between β-strand 2 and α-helix 2) faces W503, a mutation in whichconfers resistance to imidazolinones. Residues Y47 to G50 are also incontact with TPP. This loop is adjacent to P184-Q189, another turn,which connects the last strand of the β-sheet of domain 1 with aβ-strand that connects with domain 2. Within the pocket, near itsentrance, is a long region of domain 1 that interacts with acomplementary stretch of domain 2. Residues 125-129 and 133-137 ofdomain 1 and residues 304-313 of domain 2 are at the surface of thepocket. A turn consisting of T96-G100 is between loop 125-129 and TPP. Afurther stretch of domain 3 and two regions of domain 2 that line thebinding pocket are at the opposite corner of the pocket. Residues 572,575, 582, and 583 of domain 3 define the pocket surface on one side. Theremaining part of the interior of the pocket's surface is defined by FADand by a loop, L278-G282, that contacts the isoalloxazine ring of FAD.

The structural models of the AHAS protein can also be used for therational design of herbicides or AHAS inhibitors.

2. Modelling of Herbicides Into Binding Sites

Imazethapyr, the active imidazolinone in PURSUIT®, was positioned intoits proposed binding site using interactive molecular graphics (FIG. 4)and the software described above (FIG. 4). K185 was chosen as an“anchor” to interact with the charge of the carboxyl group. Theimidazolinone's NH—CO unit was placed to form hydrogen bonds to G50 andA51. This positioned the methyl substitute of imazethapyr close to V500on the backbone of the small α-helix. The isopropyl group is possiblybound by hydrophobic residues of the amino acids in the region ofresidues 125-135 that contribute to the inner surface of the pocket. Thepyridine ring is most probably “sandwiched” between A134 or F135, F507and W503. W503 also interacts with the imidazolinone ring system.

In a similar fashion, the sulfonylurea herbicides were modelled into asite that partially overlapped the described imidazolinone binding site.Overlap of sulfonylurea and imidazolinone binding sites was consistentwith competition binding experiments and with established mutant data,which show that the same mutation in maize, W503L, can confer resistanceto both herbicides. In these models, most of the known mutation sitesthat confer sulfonylurea herbicide resistance, i.e. G50, A51, K185,V500, W503, F507, are in close contact to the bound herbicides. P126 andA51 are required for keeping the K185 side chain in place by generatinga hydrophobic pore. S582, a site for specific imidazolinone resistance,is distant from the binding region and is located in the region wherethe homology is so poor that a change in the fold is expected. The FADbinding site apparently has low homology between AHAS and POX in thisregion; S582 is a residue that confers resistance in maize, and thatS582 and its adjacent residues are in close contact to the active sitepocket. It is proposed that FAD and the loop region encompassingresidues 278 to 285 move slightly away from the third domain, (downwardin FIG. 4) and that a loop that contains S582 folds into the spacebetween the helix at positions 499 to 507 and the loop at positions 278to 285. D305, another known resistance site, is close to FAD andmodulates the interaction between domains 1 and 2. M280 may either beinvolved in positioning of the helix at positions 498 to 507 or directlyin inhibitor binding. M280 and D305 could also be directly involved ininhibitor binding if domains 1 and 2 move slightly closer to each other.

3. Selection of Mutations

Specific amino acid residues are pinpointed as sites for theintroduction of mutations into the primary sequence of AHAS. These aminoacids are selected based upon their position in that if that amino acidresidue position is modified, there will be a resultant alteration (i.e.decline) in the affinity of an herbicide for the binding pocket. It isnot necessary that the mutation position reside in the binding pocket asamino acid residues outside the pocket itself can alter the pocketcharge or configuration. The selection of target sites for mutation isachieved using molecular models as described above. For exampleaccording to the model above, arginine at position 128 (designated R128in FIG. 1 using the single-letter code for amino acids) is located nearthe entrance to the substrate- and herbicide-binding pocket and has alarge degree of conformational freedom that may allow it to participatein transport of charged herbicides into the binding pocket. Therefore,this residue is substituted by alanine to remove both its charge and itslong hydrophobic side chain. (The resulting mutation is designatedR128A).

The mutations may comprise simple substitutions, which replace thewild-type sequence with any other amino acid. Alternatively, themutations may comprise deletions or additions of one or more aminoacids, preferably up to 5, at a given site. The added sequence maycomprise an amino acid sequence known to exist in another protein, ormay comprise a completely synthetic sequence. Furthermore, more than onemutation and/or more than one type of mutation may be introduced into asingle polypeptide.

4. Site-Directed Mutagenesis

The DNA encoding AHAS can be manipulated so as to introduce the desiredmutations. Mutagenesis is carried out using methods that are standard inthe art, as described in, for example, Higuchi, R., Recombinant PCR, InM. A. Innis, et al., eds, PCR Protocols: A Guide to Methods andApplications, Academic Press, pp. 177-183, 1990.

5. Expression and Purification of Variants

The mutated or variant AHAS sequence is cloned into a DNA expressionvector (see, e.g., Example 3) and is expressed in a suitable cell suchas, for example, E. coli. Preferably, the DNA encoding AHAS is linked toa transcription regulatory element, and the variant AHAS is expressed aspart of a fusion protein, for example, glutathione-S-transferase, tofacilitate purification (see Example 3 below). The variant AHAS is thenpurified using affinity chromatography or any other suitable methodknown in the art. “Purification” of an AHAS polypeptide refers to theisolation of the AHAS polypeptide in a form that allows its enzymaticactivity to be measured without interference by other components of thecell in which the polypeptide is expressed.

6. Assaying of Enzymatic Properties

The purified variant AHAS may be assayed for one or more of thefollowing three properties:

-   -   (a) specific or catalytic activity for conversion of pyruvate to        acetolactate (expressed as units/mg pure AHAS, wherein a unit of        activity is defined as 1 μmole acetolactate produced/hour), or        for condensation of pyruvate and 2-ketobutyrate to form        acetohydroxybutyrate (expressed as units/mg pure AHAS, wherein a        unit of activity is defined as 1 μmole acetohydroxybutyrate        produced/hr.;    -   (b) level of inhibition by herbicide, such as, for example,        imidazolinone (expressed as IC₅₀, the concentration at which 50%        of the activity of the enzyme is inhibited); and    -   (c) selectivity of resistance to the selected herbicide vs.        other herbicides. The selectivity index is defined as the fold        resistance of the mutant to imidazolinones relative to the        wild-type enzyme, divided by the fold resistance of the same        mutant to other herbicides also relative to the wild-type). Fold        resistance to an herbicide relative to the wild-type enzyme is        expressed as the IC₅₀ of variant, divided by the IC₅₀ of the        wild type. The selectivity index (S.I.) is thus represented by        the following equation:        ${S.I.} = \frac{{IC}_{50}\quad{of}\quad{variant}\quad{for}\quad{{{herb}.A}/{IC}_{50}}\quad{of}\quad{wild}\quad{type}\quad{for}\quad{{herb}.A}}{{IC}_{50}\quad{of}\quad{variant}\quad{for}\quad{{{herb}.B}/{IC}_{50}}\quad{of}\quad{wild}\quad{type}\quad{for}\quad{{herb}.B}}$

Suitable assay systems for making these determinations include, but arenot limited to, those described in detail in Example 4 below.

7.a. Evaluation of Suitable Variants

The enzymatic properties of variant AHAS polypeptides are compared tothe wild-type AHAS. Preferably, a given mutation results in an AHASvariant polypeptide that retains in vitro enzymatic activity towardspyruvate or pyruvate and 2-ketobutyrate, i.e., the conversion ofpyruvate to acetolactate or in the condensation of pyruvate and2-ketobutyrate to form acetohydroxybutyrate (and thus is expected to bebiologically active in vivo), while exhibiting catalytic activity thatis relatively more resistant to the selected herbicide(s) than iswild-type AHAS. Preferably, the variant AHAS exhibits:

-   -   (i) in the absence of the at least one herbicide,        -   (a) catalytic activity alone sufficient to maintain the            viability of a cell in which it is expressed; or        -   (b) catalytic activity in combination with any herbicide            resistant AHAS variant protein also expressed in the cell,            which may be the same as or different than the first AHAS            variant protein, sufficient to maintain the viability of a            cell in which it is expressed;        -   wherein the cell requires AHAS activity for viability; and    -   (ii) catalytic activity that is more resistant to the at least        one herbicide than is wild type AHAS;    -   and that is relatively more resistant to the herbicide(s) than        is wild-type AHAS.

Therefore, any one specific AHAS variant protein need not have the totalcatalytic activity necessary to maintain the viability of the cell, butmust have some catalytic activity in an amount, alone or in combinationwith the catalytic activity of additional copies of the same AHASvariant and/or the catalytic activity of other AHAS variant protein(s),sufficient to maintain the viability of a cell that requires AHASactivity for viability. For example, catalytic activity may be increasedto minimum acceptable levels by introducing multiple copies of a variantencoding gene into the cell or by introducing the gene which furtherincludes a relatively strong promoter to enhance the production of thevariant.

More resistant means that the catalytic activity of the variant isdiminished by the herbicide(s), if at all, to a lesser degree thanwild-type AHAS catalytic activity is diminished by the herbicide(s).Preferred more resistant variant AHAS retains sufficient catalytic tomaintain the viability of a cell, plant, or organism wherein at the sameconcentration of the same herbicide(s), wild-type AHAS would not retainsufficient catalytic activity to maintain the viability of the cell,plant, or organism.

Preferably the catalytic activity in the absence of herbicide(s) is atleast about 5% and, most preferably, is more than about 20% of thecatalytic activity of the wild-type AHAS in the absence of herbicide(s).Most preferred AHAS variants are more resistant to imidazolinoneherbicides than to other herbicides such as sulfonylurea-basedherbicides, though in some applications selectivity is neither needednor preferred.

In the case of imidazolinone-resistant variant AHAS, it is preferredthat the AHAS variant protein has

(i) catalytic activity in the absence of said herbicide of more thanabout 20% of the catalytic activity of said wild-type AHAS;

(ii) catalytic activity that is relatively more resistant to presence ofimidazolinone herbicides compared to wild type AHAS; and

(iii) catalytic activity that is relatively more sensitive to thepresence of sulfonylurea herbicides compared to imidazolinoneherbicides. Most preferred herbicide-resistant AHAS variants exhibit aminimum specific activity of about 20 units/mg, minimal or no inhibitionby imidazolinone, and a selectivity index ranging from about 1.3 toabout 3000 relative to other herbicides.

Without wishing to be bound by theory, it is believed that systematicand iterative application of this method to wild type or other targetAHAS protein will result in the production of AHAS variants having thedesired properties of high enzymatic activity as explained above andresistance to one or more classes of herbicides. For example, mutationof a wild-type AHAS sequence at a particular position to a given aminoacid may result in a mutant that exhibits a high degree of herbicideresistance but a significant loss of enzymatic activity towards pyruvateor pyruvate and 2-ketobutyrate. In a second application of the abovemethod, the starting or target AHAS polypeptide would then be thisvariant (in place of the wild-type AHAS). Rational design then involvessubstituting other amino acids at the originally mutated position and/oradding or deleting amino acids at selected points or ranges in theexpectation of retaining herbicide resistance but also maintaining ahigher level of enzymatic activity.

The structure-based rational design of herbicide resistant AHAS proteinsoffers many advantages over conventional approaches that rely on randommutagenesis and selection. For example, when substitution of aparticular amino acid with another requires substitution of more thanone nucleotide within the codon, the likelihood of this occurringrandomly is so low as to be impractical. By contrast, even double ortriple changes in nucleotide sequence within a codon can be easilyimplemented when suggested by a rational design approach. For example,one rationally designed mutation to confer selective imidazolinoneresistance requires a change from arginine to glutamate. Arginine isencoded by CGT, CGC, CGA, CGG, AGA, AGG, while glutamate is encoded byGAA and GAG. Since none of the arginine codons begins with GA, thismutation would require a double substitution of adjacent nucleotideswhich would occur so rarely using random mutagenesis as to beunpredictable and unrepeatable with any certainty of success. Althoughmutation frequency can be increased during random mutagenesis,alterations in nucleotide sequence would have an equal probability ofoccurring throughout the AHAS gene, in the absence of priorsite-direction of the mutations. This increases the chance of obtainingan irrelevant mutation that interferes with enzymatic activity.Similarly, it would be rare, using random mutagenesis, to find amultiple amino acid substitution, deletion, or substitution/deletionmutation that confers herbicide resistance while maintaining catalyticactivity. Deletion mutations that confer herbicide resistance would alsobe unlikely using a random mutagenesis approach. Deletions would need tobe limited to small regions and would have to occur in triplets so as toretain the AHAS reading frame in order to retain enzymatic activity.

However, with a rational structure-based approach, double amino acidsubstitution and/or deletion mutations are relatively easily achievedand precisely targeted. Furthermore, different mutagens used in randommutagenesis create specific types of mutations. For example, sodiumazide creates point substitution mutations in plants, while radiationtends to create deletions. Accordingly, two mutagenesis protocols wouldhave to be employed to obtain a multiple combinationsubstitution/deletion.

Finally, the present structure-based method for rational design ofherbicide-resistant AHAS variants allows for iterative improvement ofherbicide resistance mutations, a step that is not facilitated by randommutagenesis. Identification of a mutation site for herbicide resistanceby random mutagenesis may offer little, if any, predictive value forguiding further improvements in the characteristics of the mutant. Thepresent structure-based approach, on the other hand, allows improvementsto be implemented based on the position, environment, and function ofthe amino acid position in the structural model.

The iterative improvement method also allows the independentmanipulation of three important properties of AHAS: level of resistance,selectivity of resistance, and catalytic efficiency. For example,compensatory mutations can be designed in a predictive manner. If aparticular, mutation has a deleterious effect on the activity of anenzyme, a second compensatory mutation may be used to restore activity.For example, a change in the net charge within a domain when a chargedresidue is introduced or lost due to a mutation can be compensated byintroducing a second mutation. Prediction of the position and type ofresidue(s) to introduce, delete, or substitute at the second site inorder to restore enzymatic activity requires a knowledge ofstructure-function relationships derived from a model such as thatdescribed herein.

7.b. Design of Non-Peptide Herbicides or AHAS Inhibitors

A chemical entity that alters and may fit into the active site of thetarget-protein or bind in any position where it could inhibit activitymay be designed by methods known in the art, such as, for example,computer design programs that assist in the design of compounds thatspecifically interact with a receptor site.

An example of such a program is LUDI (Biosym Technologies—San Diego,Calif.) (see also, Lam, et al., Science 263:380, 1994; Thompson, et al.,J. Med. Chem., 37:3100, 1994).

The binding pocket and particularly the amino acid residues that havebeen identified as being involved as inhibitor binding can be used asanchor points for inhibitor design.

The design of site-specific herbicides is advantageous in the control ofweed species that may spontaneously develop herbicide resistance in thefield, particularly due to mutations in the AHAS gene.

Herbicide-Resistant AHAS Variants: DNA, Vectors, and Polypeptides

The present invention also encompasses isolated DNA molecules encodingvariant herbicide-resistant AHAS polypeptides. Genes encoding AHASpolypeptides according to the present invention may be derived from anyspecies and preferably a plant species, and mutations conferringherbicide resistance may be introduced at equivalent positions withinany of these AHAS genes. The equivalence of a given codon position indifferent AHAS genes is a function of both the conservation of primaryamino acid sequence and its protein and the retention of similarthree-dimensional structure. For example, FIG. 5 illustrates the highdegree of sequence homology between AHAS polypeptides derived fromdifferent plant species. These AHAS polypeptides exhibit at least about60 to about 70% overall homology. Without wishing to be bound by theory,it is believed that in regions of the polypeptide having a highlyconserved sequence, the polypeptide chain conformation will also bepreserved. Thus, it is possible to use an AHAS-encoding sequence fromone species for molecular modelling, to introduce mutations predictivelyinto an AHAS gene from a second species for initial testing anditerative improvement, and finally, to introduce the optimized mutationsinto AHAS derived from yet a third plant species for expression in atransgenic plant.

In one series of embodiment, these AHAS DNAs encode variants of an AHASpolypeptide and preferably of the maize AHAS polypeptide of FIG. 1 (SEQID NO:1) in which the polypeptide is modified by substitution at ordeletion preceding or following one or more of FIG. 1 (SEQ ID NO:1)amino acid residues P48, G49, S52, M53, E54, A84, A95, T96, S97, G98,P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133, F135,Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278, G279,H281, G282, T283, V284, G300, V301, R302, F303, D304, R306, V307, T308,G309, K310, I311, E312, A313, F314, A315, S316, R317, A318, K319, I320,E329, I330, K332, N333, K334, Q335, T404, G413, V414, G415, Q416, H417,Q418, M419, W420, A421, A422, L434, S435, S436, A437, G438, L439, G440,A441, M442, G443, D467, G468, S469, L471, N473, L477, M479, Q495, H496,L497, G498, M499, V501, Q502, Q504, D505, R506, Y508, K509, A510, N511,R521, A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578,M579, I580, P581, G583, G584, functional equivalents of any of theforegoing; insertions or deletions between FIG. 1 (SEQ ID NO:1) Q124 andH150 or functional equivalents thereof; insertions or deletions betweenFIG. 1 (SEQ ID NO:1) G300 and D324 or functional equivalents thereof;and any combination of any of the foregoing thereof.

The mutations, whether introduced into the polypeptide of FIG. 1 (SEQ IDNO:1) or at equivalent positions in another plant AHAS gene, maycomprise alterations in DNA sequence that result in a simplesubstitution of any one or more other amino acids or deletions of up to5 ammo acid residues proceeding or up to 5 amino acids residuesfollowing any of the residues listed above. Suitable amino acidsubstitutes include, but are not limited to, naturally occurring aminoacids.

Alternatively, the mutations may comprise alterations in DNA sequencesuch that one or more amino acids are added or deleted in frame at theabove positions. Preferably, additions comprise about 3 to about 30nucleotides, and deletions comprise about 3 to about 30 nucleotides.Furthermore, a single mutant polypeptide may contain more than onesimilar or different mutation.

The present invention encompasses DNA and corresponding RNA sequences,as well as sense and antisense sequences. Nucleic acid sequencesencoding AHAS polypeptides may be flanked by natural AHAS regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-noncoding regions, andthe like. Furthermore, the nucleic acids can be modified to alterstability, solubility, binding affinity and specificity. For example,variant AHAS-encoding sequences can be selectively methylated. Thenucleic acid sequences of the present invention may also be modifiedwith a label capable of providing a detectable signal, either directlyor indirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

The invention also provides vectors comprising nucleic acids encodingAHAS variants. A large number of vectors, including plasmid and fungalvectors, have been described for expression in a variety of eukaryoticand prokaryotic hosts. Advantageously, vectors may also include apromotor operably linked to the AHAS encoding portion. The encoded AHASmay be expressed by using any suitable vectors and host cells, usingmethods disclosed or cited herein or otherwise known to those skilled inthe relevant art. Examples of suitable vectors include withoutlimitation pBIN-based vectors, pBluescript vectors, and pGEM vectors.

The present invention also encompasses both variant herbicide-resistantAHAS polypeptides or peptide fragments thereof. As explained above, thevariant AHAS polypeptides may be derived from the maize polypeptideshown in FIG. 1 or from any plant or microbial AHAS polypeptide,preferably plant AHAS polypeptide. The polypeptides may be furthermodified by, for example, phosphorylation, sulfation, acylation,glycosylation, or other protein modifications. The polypeptides may beisolated from plants, or from heterologous organisms or cells(including, but not limited to, bacteria, yeast, insect, plant, andmammalian cells) into which the gene encoding a variant AHAS polypeptidehas been introduced and expressed. Furthermore, AHAS polypeptides may bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly, including radioisotopes, fluorescent compounds,and the like.

Chemical-resistant Plants and Plants Containing Variant AHAS Genes

The present invention encompasses transgenic cells, including, but notlimited to seeds, organisms, and plants into which genes encodingherbicide-resistant AHAS variants have been introduced. Non-limitingexamples of suitable recipient plants are listed in Table 1 below:

TABLE 1 RECIPIENT PLANTS COMMON NAME FAMILY LATIN NAME Maize GramineaeZea mays Maize, Dent Gramineae Zea mays dentiformis Maize, FlintGramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microspermaMaize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea maysamyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, WaxyGramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum speltaWheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticumturgidum Wheat, Large Pooideae Triticum spelta Spelt Wheat, PolishPooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidumWheat, Pooideae Triticum monococcum Singlegrained Wheat, Small PooideaeTriticum monococcum Spelt Wheat, Soft Pooideae Triticum aestivum RiceGramineae Oryza sativa Rice, American Gramineae Zizania aquatica WildRice, Australian Gramineae Oryza australiensis Rice, Indian GramineaeZizania aquatica Rice, Red Gramineae Oryza glaberrima Rice, TuscaroraGramineae Zizania aquatica Rice, West Gramineae Oryza glaberrima AfricanBarley Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeumirregulare Intermediate, also Irregular Barley, Pooideae Hordeumspontaneum Ancestral Tworow Barley. Beardless Pooideae Hordeumtrifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley,fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed PooideaeHordeum vulgare hexastichon Barley, Tworowed Pooideae Hordeum distichonCotton, Abroma Dicotyledoneae Abroma augusta Cotton, American MalvaceaeGossypium hirsutum Upland Cotton, Asiatic Malvaceae Gossypium arboreumTree, also Indian Tree Cotton, Brazilian, Malvaceae Gossypium barbadensealso, Kidney, brasiliense and, Pernambuco Cotton, Levant MalvaceaeGossypium herbaceum Cotton, Long Silk, Malvaceae Gossypium barbadensealso Long Staple, Sea Island Cotton, Mexican, Malvaceae Gossypiumhirsutum also Short Staple Soybean, Soya Leguminosae Glycine max Sugarbeet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plantArenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, CherrySolanaceae Lycopersicon esculentum cerasiforme Tomato, Common SolanaceaeLycopersicon esculentum commune Tomato, Currant Solanaceae Lycopersiconpimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato,Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersiconesculentum pyriforme Tomato, Tree Solanaceae Cyphomandra betacea PotatoSolanaceae Solanum tuberosum Potato, Spanish, Convolvulaceae Ipomoeabatatas Sweet potato Rye, Common Pooideae Secale cereale Rye, MountainPooideae Secale montanum Pepper, Bell Solanaceae Capsicum annuum grossumPepper, Bird, also Solanaceae Capsicum annuum Cayenne, Guinea minimumPepper, Bonnet Solanaceae Capsicum sinense Pepper, Bullnose, SolanaceaeCapsicum annuum grossum also Sweet Pepper, Cherry Solanaceae Capsicumannuum cerasiforme Pepper, Cluster, Solanaceae Capsicum annuum also RedCluster fasciculatum Pepper, Cone Solanaceae Capsicum annuum conoidesPepper, Goat, Solanaceae Capsicum frutescens also Spur Pepper, LongSolanaceae Capsicum frutescens longum Pepper, Solanaceae Capsicum annuumOranamental Red, abbreviatum also Wrinkled Pepper, Tabasco SolanaceaeCapsicum annuum conoides Red Lettuce, Garden Compositae Lactuca sativaLettuce, Asparagus, Compositae Lactuca sativa also Celery asparaginaLettuce, Blue Compositae Lactuca perennis Lettuce, Blue, CompositaeLactuca pulchella also Chicory Lettuce, Cabbage, Compositae Lactucasativa capitata also Head Lettuce, Cos, Compositae Lactuca sativalongifolia also Longleaf, Romaine Lettuce, Crinkle, Compositae Lactucasativa crispa also Curled, Cutting, Leaf Celery Umbelliferae Apiumgraveolens dulce Celery, Blanching, Umbelliferae Apium graveolens dulcealso Garden Celery, Root, Umbelliferae Apium graveolens rapaceum alsoTurniprooted Eggplant, Garden Solanaceae Solanum melongena SorghumSorghum All crop species Alfalfa Leguminosae Medicago sativum CarrotUmbelliferae Daucus carota sativa Bean, Climbing Leguminosae Phaseolusvulgaris vulgaris Bean, Sprouts Leguminosae Phaseolus aureus Bean,Brazilian Leguminosae Canavalia ensiformis Broad Bean, Broad LeguminosaeVicia faba Bean, Common, Leguminosae Phaseolus vulgaris also French,White, Kidney Bean, Egyptian Leguminosae Dolichos lablab Bean, Long,also Leguminosae Vigna sesquipedalis Yardlong Bean, Winged LeguminosaePsophocarpus tetragonolobus Oat, also Common, Avena Sativa Side, TreeOat, Black, also Avena Strigosa Bristle, Lopsided Oat, Bristle AvenaPea, also Garden, Leguminosae Pisum, sativum sativum Green, ShellingPea, Blackeyed Leguminosae Vigna sinensis Pea, Edible Podded LeguminosaePisum sativum axiphium Pea, Grey Leguminosae Pisum sativum speciosumPea, Winged Leguminosae Tetragonolobus purpureus Pea, WrinkledLeguminosae Pisum sativum medullare Sunflower Compositae Helianthusannuus Squash, Autumn, Dicotyledoneae Cucurbita maxima Winter Squash,Bush, Dicotyledoneae Cucurbita pepo also Summer melopepo Squash, TurbanDicotyledoneae Cucurbita maxima turbaniformis Cucumber DicotyledoneaeCucumis sativus Cucumber, African, Momordica charantia also BitterCucumber, Squirting, Ecballium elaterium also Wild Cucumber, WildCucumis anguria Poplar, California Woody-Plant Populus trichocarpaPoplar, European Populus nigra Black Poplar, Gray Populus canescensPoplar, Lombardy Populus italica Poplar, Silverleaf, Populus alba alsoWhite Poplar, Western Populus trichocarpa Balsam Tobacco SolanaceaeNicotiana Arabidopsis Cruciferae Arabidopsis thaliana Thaliana TurfgrassLolium Turfgrass Agrostis Other families of turfgrass Clover Leguminosae

Expression of the variant AHAS polypeptides in transgenic plants confersa high level of resistance to herbicides including, but not limited to,imidazolinone herbicides such as, for example, imazethapyr (PURSUIT®),allowing the use of these herbicides during cultivation of thetransgenic plants.

Methods for the introduction of foreign genes into plants are known inthe art. Non-limiting examples of such methods include Agrobacteriuminfection, particle bombardment, polyethylene glycol (PEG) treatment ofprotoplasts, electroporation of protoplasts, microinjection,macroinjection, tiller injection, pollen tube pathway, dry seedimbibition, laser perforation, and electrophoresis. These methods aredescribed in, for example, B. Jenes et al., and S. W. Ritchie et al. InTransgenic Plants, Vol. 1, Engineering and Utilization, ed. S.-D. Kung,R. Wu, Academic Press, Inc., Harcourt Brace Jovanovich 1993; and L.Mannonen et al., Critical Reviews in Biotechnology, 14:287-310, 1994.

In a preferred embodiment, the DNA encoding a variant AHAS is clonedinto a DNA vector containing an antibiotic resistance marker gene, andthe recombinant AHAS DNA-containing plasmid is introduced intoAgrobacterium tumefaciens containing a Ti plasmid. This “binary vectorsystem” is described in, for example, U.S. Pat. No. 4,490,838, and in Anet al., Plant Mol. Biol. Manual A3:1-19 (1988). The transformedAgrobacterium is then co-cultivated with leaf disks from the recipientplant to allow infection and transformation of plant cells. Transformedplant cells are then cultivated in regeneration medium, which promotesthe formation of shoots, first in the presence of the appropriateantibiotic to select for transformed cells, then in the presence ofherbicide. In plant cells successfully transformed with DNA encodingherbicide-resistant AHAS, shoot formation occurs even in the presence oflevels of herbicide that inhibit shoot formation from non-transformedcells. After confirming the presence of variant AHAS DNA using, forexample, polymerase chain reaction (PCR) analysis, transformed plantsare tested for their ability to withstand herbicide spraying and fortheir capabilities for seed germination and root initiation andproliferation in the presence of herbicide.

Other Applications

The methods and compositions of the present invention can be used in thestructure-based rational design of herbicide-resistant AHAS variants,which can be incorporated into plants to confer selective herbicideresistance on the plants. Intermediate variants of AHAS (for example,variants that exhibit sub-optimal specific activity but high resistanceand selectivity, or the converse) are useful as templates for the designof second-generation AHAS variants that retain adequate specificactivity and high resistance and selectivity.

Herbicide resistant AHAS genes can be transformed into crop species insingle or multiple copies to confer herbicide resistance. Geneticengineering of crop species with reduced sensitivity to herbicides can:

(1) Increase the spectrum and flexibility of application of specificeffective and. environmentally benign herbicides such as imidazolinoneherbicides;

(2) Enhance the commercial value of these herbicides;

(3) Reduce weed pressure in crop fields by effective use of herbicideson herbicide resistant crop species and a corresponding increase inharvest yields;

(4) Increase sales of seed for herbicide-resistant plants;

(5) Increase resistance to crop damage from carry-over of herbicidesapplied in a previous planting;

(6) Decrease susceptibility to changes in herbicide characteristics dueto adverse climate conditions; and

(7) Increase tolerance to unevenly or mis-applied herbicides.

For example, transgenic AHAS variant protein containing plants can becultivated. The crop can be treated with a weed controlling effectiveamount of the herbicide to which the AHAS variant transgenic plant isresistant, resulting in weed control in the crop without detrimentallyaffecting the cultivated crop.

The DNA vectors described above that encode herbicide-resistant AHASvariants can be further utilized so that expression of the AHAS variantprovides a selectable marker for transformation of cells by the vector.The intended recipient cells may be in culture or in situ, and the AHASvariant genes may be used alone or in combination with other selectablemarkers. The only requirement is that the recipient cell is sensitive tothe cytotoxic effects of the cognate herbicide. This embodiment takesadvantage of the relative low cost and lack of toxicity of, for example,imidazolinone-based herbicides, and may be applied in any system thatrequires DNA-mediated transformation.

Exemplification with respect to Preferred Embodiments

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLE 1 Design of Herbicide-Resistant AHAS Variants

Residues located close to the proposed herbicide binding site of themodel described in detail above were selected for mutagenesis in orderto design an active AHAS polypeptide with decreased herbicide bindingcapacity. Each site at the surface of the pocket was considered in termsof potential interactions with other residues in the pocket, as well aswith cofactors and herbicides. For example, addition of positivelycharged residue(s) is expected to interfere with the charge distributionwithin the binding site, resulting in a loss in affinity of binding of anegatively-charged herbicide.

Three residues were identified as most useful targets for mutagenesis:

(1) F135 was believed to interact with both the isoalloxazine ring ofFAD and with the aromatic group of the herbicides. In accordance withthe strategy of introducing more charged residues into the bindingpocket, this residue was changed to arginine.

(2) M53 contacts helix 498-507. This helix contains known herbicideresistance mutation sites and is also implicated in TPP binding.Furthermore, substitution of glutamic acid at position 53 was believedto favor an interaction with K185, reducing the affinity of K185 for thecarboxylate group of imazethapyr.

(3) R128 is located near the entrance to the pocket, where it wasbelieved to be involved in the initial transport of charged herbicidesinto the binding pocket. This residue was changed to alanine to removeboth its charge and its long hydrophobic side chain.

EXAMPLE 2 Site-directed Mutagenesis of AHAS to ProduceHerbicide-resistant Variants

The Arabidopsis AHAS gene was inserted in-frame to the 3′ end of thecoding region of the glutathione S-transferase gene in the pGEX-2Tvector (Pharmacia). Construction of the vector in this manner maintainedthe six amino acid thrombin recognition sequence at the junction of theexpressed glutathione-S-transferase (GST)/AHAS fusion protein. Thrombindigestion of the expressed fusion protein results in an AHAS proteinwith an N-terminal starting position at the end of the transit peptideat a putative transit peptide processing site, with a residualN-terminal glycine derived from the thrombin recognition site. The finalamino terminus of the cleaved AHAS protein consists ofGly-Ser-Ser-Ile-Ser. Site-directed mutations were introduced into theAHAS gene in this vector.

Site-directed mutations were constructed according to the PCR method ofHiguchi (Recombinant PCR. In M A Innis, et al. PCR Protocols: A Guide toMethods and Applications, Academic Press, San Diego, pp. 177-183, 1990).Two PCR products, each of which overlap the mutation site, wereamplified. The primers in the overlap region contained the mutation. Theoverlapping PCR amplified fragments were combined, denatured, andallowed to re-anneal together, producing two possible heteroduplexproducts with recessed 3′-ends. The recessed 3′-ends were extended byTaq DNA polymerase to produce a fragment that was the sum of the twooverlapping PCR products containing the desired mutation. A subsequentre-amplification of this fragment with only the two “outside” primersresulted in the enrichment of the full-length product. The productcontaining the mutation was then re-introduced into the Arabidopsis AHASgene in the pGEX-2T vector.

EXAMPLE 3 Expression and Purification of AHAS Variants

A. Methods

E. Coli (DH5α) cells transformed with the pGEX-2T vector containingeither the maize wild type AHAS gene (vector designation pAC751), theArabidopsis Ser653Asn mutant, or the Arabidopsis Ile401Phe mutant weregrown overnight in LB broth containing 50 μg/mL ampicillin. Theovernight culture of E. coli was diluted 1:10 in 1 L LB, 50 μg/mLampicillin, and 0.1% v/v antifoam A. The culture was incubated at 37° C.with shaking until the OD₆₀₀ reached approximately 0.8.Isopropylthiogalactose (IPTG) was added to a final concentration of 1 mMand the culture was incubated for 3 more hours.

Cells were harvested by centrifugation at 8,670×g for 10 minutes in aJA-10 rotor and resuspended in {fraction (1/100)}th of the originalculture volume in MTPBS (16 mM Na₂HPO₄, 4 mM NaH₂PO₄, 150 mM NaCl, pH7.3). Triton X-100 and lysozyme were added to a final concentration of1% v/v and 100 μg/mL, respectively. Cells were incubated at 30° C. for15 minutes cooled to 4° C. on ice, and were lysed by sonication for 10seconds at level 7 with a Branson Sonifier Cell Disrupter equipped witha microtip probe. The cell free extract was centrifuged at 35,000 ×g for10 min. at 4° C. The supernatant was decanted and the centrifugationstep was repeated.

Purification of expressed fusion proteins was performed as modified fromSmith and Johnson (Gene 67:31-40, 1988). The supernatant was warmed toroom temperature and was passed through a 2 mL column ofglutathione-agarose beads (sulfur linkage, Sigma) equilibrated in MTPBS.The column was subsequently washed with MTPBS at room temperature untilthe A₂₈₀ of eluant matched that of MTPBS. The fusion protein was theneluted using a solution containing 5 mM reduced glutathione in 50 mMTris HCL, pH 8.0. The eluted fusion protein was treated withapproximately 30 NIH units of thrombin and dialyzed against 50 mMcitrate pH 6.5 and 150 mM NaCl.

The fusion protein was digested overnight at room temperature. Digestedsamples were dialyzed against MTPBS and passed twice through aglutathione-agarose column equilibrated in MTPBS to remove the releasedglutathione transferase protein. The protein fraction that did not bindto the column was collected and was concentrated by ultrafiltration on aYM10 filter (Amicon). The concentrated sample was loaded onto a 1.5×95cm Sephacryl S-100 gel filtration column equilibrated in gel filtrationbuffer (50 mM HEPES, 150 mM NaCl, pH 7.0). Two mL fractions werecollected at a flow rate of 0.14 mL/min. Enzyme stability was tested bystorage of the enzyme at 4° C. in gel filtration buffer with theaddition of 0.02% sodium azide and in the presence or absence of 2 mMthiamine pyrophosphate and 100 μM flavin adenine dinucleotide (FAD).

B. Results

E. coli transformed with the pAC751 plasmid containing the wide-typeAHAS gene fused downstream and in-frame with the GST gene expressed a 91kD protein when induced with IPTG. The 91 kD protein exhibited thepredicted molecular mass of a GST/AHAS fusion protein (the sum of 26 kDand 65 kD, respectively). When the cell free extract of DH5α/pAC751 waspassed through a glutathione-agarose affinity gel, washed, and elutedwith free glutathione it yielded a preparation enriched in the 91 kDprotein (FIG. 6, lane C). The six amino acid thrombin recognition siteengineered in the junction of GST and AHAS was successfully cleaved bythrombin (FIG. 6, lane D). The cleaved fusion protein preparationconsisted of the expected 26 kD GST protein and the 65 kD maize AHASprotein. Maize AHAS was purified to homogeneity by a second pass throughthe glutathione-agarose column to affinity subtract GST and subjected toa final Sephacryl S-100 gel filtration step to eliminated thrombin (FIG.6, lane E). The 65 kD protein is recognized on western blots by amonoclonal antibody raised against a maize AHAS peptide.

Purified wild type maize AHAS was analyzed by electrospray massspectrometry and was determined to have a molecular mass of 64,996daltons (data not shown). The predicted mass, as calculated from thededuced amino acid sequence of the gene inserted into the pGEX-2Tvector, is 65,058. The 0.096% discrepancy between the empiricallydetermined and predicted mass was within tuning variability of the massspectrometer. The close proximity of the two mass determinationssuggests that there were no misincorporated nucleotides duringconstruction of the expression vector, nor any post-translationalmodifications to the protein that would cause gross changes in molecularmass. Moreover, the lack of spurious peaks in the preparation ofpurified enzyme indicated that the sample was free of contamination.

EXAMPLE 4 Enzymatic Properties of AHAS Variants

The enzymatic properties of wild-type and variant AHAS produced in E.coli were measured by a modification of the method of Singh et al.(Anal. Biochem 171:173-179, 1988) as follows:

A reaction mixture containing 1× AHAS assay buffer (50 mM HEPES pH 7.0,100 mM pyruvate, 10 MM MgCl₂, 1 mM thiamine pyrophosphate (TPP), and 50μM flavin adenine dinucleotide (FAD)) was obtained either by dilution ofenzyme in 2× assay buffer or by addition of concentrated enzyme to 1×AHAS assay buffer. All assays containing imazethapyr and associatedcontrols contained a final concentration of 5% DMSO due to addition ofimazethapyr to assay mixtures as a 50% DMSO solution. Assays wereperformed in a final volume of 250 μL at 37° C. in microtiter plates.After allowing the reaction to proceed for 60 minutes, acetolactateaccumulation was measured calorimetrically as described by Singh et al.,Anal. Biochem 171:173-179, 1988.

Maize AHAS expressed and purified from pAC751 as described in Example 3above is active in the conversion of pyruvate to acetolactate. Full AHASactivity is dependent on the presence of the cofactors FAD and TPP inthe assay medium. No activity was detected when only FAD was added tothe assay medium. The activity of the purified enzyme with TPP only, orwith no cofactors, was less than 1% of the activity detected in thepresence of both TPP and FAD. Normally, AHAS present in crude plantextracts is very labile, particularly in the absence of substrate andcofactors. In contrast, the purified AHAS from the bacterial expressionsystem showed no loss in catalytic activity when stored for one month at4° C. in 50 mM HEPES pH 7.0, 150 mM NaCl, 0.02% NaN₃ in the presence orabsence of FAD and TPP. Furthermore, no degradation products werevisible from these stored preparations when resolved in SDS-PAGE gels.

The specific activities of wild-type AHAS and the M124E, R199A, andF206R variants are shown in Table 2 below. As determined from thealignment in FIG. 5, the M124E mutation in Arabidopsis AHAS is theequivalent of the maize M53E mutation, the R199A mutation in Arabidopsisis the equivalent of the maize R128A mutation, and the F206R mutation inArabidopsis is the equivalent of the maize F135R mutation. The mutationsdesigned in the maize AHAS structural model were used to identify theequivalent amino acid in the dicot Arabidopsis AHAS gene and wereincorporated and tested in the Arabidopsis AHAS gene. This translationand incorporation of rationally designed herbicide mutations into thedicot Arabidopsis AHAS gene can facilitate evaluation of herbicideresistance in plants of a dicot species.

TABLE 2 SPECIFIC ACTIVITY % Catalytic Activity as Specific ActivityCompared to Wild Type Wild-Type 99.8 100 Met124Glu 9.15 9.16 Arg199Ala86.3 86.5 Phe206Arg 5.07 5.1

The R199A mutation maintains a high level of catalytic activity (Table2) while exhibiting a significant level of resistance to imazethapyr(FIG. 7). Notably, this variant retains complete sensitivity tosulfonylureas (FIG. 8). Thus, this variant fulfills the criteria of highspecific activity and selective herbicide resistance. By contrast, theM124E substitution resulted in almost complete resistance to imazethapyr(FIG. 7) but also exhibited severely reduced catalytic activity (Table2). Relative to imidazolinone resistance, this variant exhibits greatersensitivity to sulfonylurea (FIG. 8), suggesting that this residue is agood candidate for creating a mutation that confers selectiveresistance. Substitution of an amino acid other than glutamic acid mayhelp to maintain catalytic activity. The F206R substitution yieldedsimilar results to those observed with M124E variant, but lackedselectivity in resistance.

EXAMPLE 5 Iterative Improvement of AHAS Herbicide-Resistant VariantUsing a Rational Design Approach

Changing residue 124 in AHAS from Met to Glu as described in Example 4above conferred imidazolinone resistance but also reduced enzymaticactivity to 9.2% of the wild type value. The model of the maize AHASstructure described above suggested that Met53 (equivalent to theArabidopsis Met124 residue) interacts with a series of hydrophobicresidues on the face of an α-helix that is derived from a separatesubunit but are in close proximity to Met53. Thus, the hydrophobicinteraction between Met53 and the residues on the helix may stabilizeboth subunit/subunit association and the conformation of the activesite. It was believed that the substitution of the hydrophobic Metresidue with a charged glutamate residue most probably destabilizes theinter-subunit hydrophobic interaction and results in a loss of catalyticactivity.

Based on this structure/function analysis, the activity of the originalArabidopsis Met124Glu (equivalent to maize Met53Glu) mutant enzyme wasthen iteratively improved by substituting a more hydrophobic amino acid(Ile) at this position. The hydrophobic nature of the Ile side chainresulted in restoration of activity to wild type levels (specificactivity of 102, equivalent to 102% of the wild-type activity) , but thegreater bulk of the Ile side chain was still able to maintain asignificant level of imidazolinone resistance (FIG. 9).

By comparison, substitution of a histidine residue at this positionresulting in an AHAS variant exhibiting a specific activity of 42.5,equivalent to 42.6% of the wild-type activity. This mutant, nonetheless,exhibited a high degree of resistance to PURSUIT® (FIG. 10).

EXAMPLE 6 Iterative Improvement of AHAS Herbicide-Resistant VariantUsing a Rational Design Approach

Another example of iterative refinement using the methods of the presentinvention involves the Arg128Als variant. The structural model of maizeAHAS suggested that the Arg128 residue, which resides at the lip of theherbicide binding pocket, contributes to channeling charged substratesand herbicides into the herbicide binding pocket and into the activesite. The Arg 128 residue is distant from the TPP moiety, which bindsthe initial pyruvate molecule in the reaction mechanism of AHAS,explaining why the substitution of Arabidopsis AHAS Arg199 (theequivalent to maize Arg128) to alanine had little effect on thecatalytic activity of the enzyme. The structural model further indicatedthat a more radical change could be made at this position to raise thelevel of resistance while maintaining high levels of catalytic activity.On this basis, an iterative improvement of the mutation was made tosubstitute the positively charge arginine residue with a negativelycharged glutamate residue. The enzyme thus mutated had improved levelsof resistance to PURSUIT® while maintaining high levels of activity(specific activity of 114, equivalent to 114% of the wild-type activity)(FIG. 11).

EXAMPLE 7 Interchangeability of AHAS Derived From Different Species inStructure-Based Rational Design of Herbicide-Resistant AHAS Variants

A structural model of the three-dimensional structure of AHAS is builtwith a monocot AHAS sequence such as that derived from maize, asdescribed above. To introduce mutations into AHAS derived from a dicotspecies such as Arabidopsis, the sequences of AHAS derived from themonocot and dicot species are aligned using the GAP and PILEUP programs(Genetics Computer Group, 575 Sequence Drive, Madison, Wis. 53711).Equivalent positions are determined from the computer-generatedalignment. The mutations are then introduced into the dicot AHAS gene asdescribed above. Following expression of the mutant AHAS protein in E.coli and assessment of its biochemical properties (i.e., specificactivity and resistance to herbicides), the mutant gene is introducedinto a dicot plant by plant transformation methods as described above.

EXAMPLE 8 Production of Herbicide-Resistant Plants by Transformationwith Rationally Designed AHAS Genes

DNA Constructs:

Rationally designed AHAS variant genes contained within E. coliexpression vectors were used as a source of DNA restriction fragments toreplace the equivalent restriction fragment in a Arabidopsis AHAS gene.This gene is present in a 5.5 kb genomic DNA fragment which alsocontains the Arabidopsis AHAS promoter, the Arabidopsis AHAS terminationsequence and 5′- and 3′-flanking DNA. After DNA sequencing through themutation sites was performed to confirm the presence of the propermutation, the entire 5.5 kb fragment from each plasmid was inserted intoa pBIN based plant transformation vector (Mogen, Leiden, Netherlands).The plant transformation vector also contains the neomycinphosphotransferase II (nptII) kanamycin resistance gene driven by the35S cauliflower mosaic virus promoter. The final vector construct isdisplayed in FIG. 12. Vectors containing Arabidopsis AHAS genes withMet124Ile, Met124His, and Arg199Glu mutations (corresponding toMet53Ile, Met53His, and Arg128Glu mutations in the maize AHAS sequenceas shown in FIG. 1) were labeled pJK002, pJK003, and pJk004,respectively.

Each of these vectors was transformed into Agrobacterium tumefaciensstrain LBA4404 (R&D Life Technologies, Gaithersburg, Md.) using thetransformation method described in An et al., Plant Mol. Biol. ManualA3:1-19 (1988).

Plant Transformation:

Leaf disc transformation of Nicotiana tabacum cv. Wisconsin 38 wasperformed as described by Horsch et al. (Science, 227: 1229-1231, 1985)with slight modifications. Leaf discs were cut from plants grown understerile conditions and co-cultivated upsidedown in Murashige Skoog media(Sigma Chemical Co., St. Louis, Mo.) for 2-3 days at 25° C. in darknesswith Agrobacterium tumefaciens strains containing plasmids pJK002,pJK003, or pJK004. The discs were blotted dry and transferred toregeneration Mutashige Skoog medium with B5 vitamins containing 1 mg/Lbenzyladenine and 0.1 mg/l 1-Napthyl Acetic Acid, 100 mg/L kanamycin,and 500 mg/L cefotaxime (all obtained from Sigma).

Initially, transformants were selected by kanamycin resistance conferredby the nptII gene present in the transformation vector. Shoots derivedfrom the leaf discs were excised and placed on fresh Murashige Skooghormone free media containing cefotaxime and kanamycin.

In Vivo Herbicide Resistance

Kanamycin-resistant tobacco shoots were transferred to medium containinga 0.25 μM imazethapyr. At this concentration of the imidazolinoneherbicide, non-transformed tobacco shoots (containing endogenouswild-type AHAS) were not able to initiate root formation. By contrast,root initiation and growth were observed from tobacco shoots transformedwith each of the mutant AHAS genes. Roots developed from shootstransformed with the Met124Ile and Arg199Glu mutant genes along withwild type are shown in FIG. 1. Furthermore, plants transformed with theMet124Ile or Arg199Glu mutant genes were resistant to spraying withtwice the field rate (100 g/ha) of imazethapyr (FIG. 13). The patternsof root growth in transformed vs. non-transformed plants in the presenceof herbicide, as well as the behavior after herbicide spraying suggestthat expression of the rationally designed herbicide resistance genesconfers herbicide resistance in vivo.

Detection of the Rationally Designed Genes in Herbicide ResistantTobacco

Genomic DNA was isolated from the AHAS-transformed tobacco plants, andthe presence of the Arabidopsis AHAS variant genes was verified by PCRanalysis. Differences between the nucleotide sequences of theArabidopsis AHAS gene and the two tobacco AHAS genes were exploited todesign PCR primers that amplify only the Arabidopsis gene in a tobaccogenomic DNA background. The rationally designed herbicide resistancegenes were detected, as shown by amplification of a DNA fragment of theproper size, in a majority of the herbicide resistant plants. No PCRsignal was seen from non-transformed tobacco plants.

Segregation of Transformed AHAS Genes:

To monitor segregation of rationally designed AHAS genes in transformedplants, germination tests were performed. Seeds were placed inhormone-free Murashige-Skoog medium containing up to 2.5 μM PURSUIT® and100 μM kanamycin. The seedlings that resulted were visually scored forresistance or susceptibility to the herbicide.

Since tobacco plants are diploid, it would be expected that the progenyof self-pollinated plants should segregate 3:1 resistant:susceptible,reflecting the existence of 1 seedling homozygous for the resistant AHASgene, 2 seedlings heterozygous for the resistant AHAS gene, and 1seedling lacking a resistant AHAS gene.

The results indicate that resistant AHAS genes are segregating in theexpected 3:1 ratio, supporting the conclusion that herbicide resistanceis conferred by a single, dominant copy of a rationally designed AHASgene.

These results indicate that rational design of herbicide-resistant AHASgenes can be used to produce plants that exhibit herbicide resistantgrowth in vivo.

EXAMPLE 9 Production of Plants Cross-Resistant to Different Herbicidesby Transformation with Rationally Designed AHAS Genes

Tobacco plants transformed with rationally designed AHAS genes asdescribed in Example 8 above were also tested for cross-resistance toanother herbicide, CL 299,263 (also known as imazamox). Germinationtests were performed on seeds harvested from the primary transformantscontaining the Met124Ile, Met124His, and Arg199Glu Arabidopsis AHASvariant genes, in the absence or presence of 2.5 μM CL 299,263 (FIG.15). This concentration of the herbicide causes severe stunting andbleaching of wild-type tobacco plants. Tobacco plants transformed withthe Met124His AHAS gene showed the greatest level of resistance (FIG.15). Arg199Glu transformants showed an intermediate level of resistance,while Met124Ile showed little resistance (FIG. 15).

All patents, applications, articles, publications, and test methodsmentioned above are hereby incorporated by reference.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

1. A variant plant acetohydroxy acid synthase (AHAS) protein comprisingat least one mutation at an amino acid residue corresponding to aminoacid G583 of SEQ ID NO: 1, wherein said variant plant AHAS protein ismore resistant to an herbicide than the wild-type plant AHAS protein. 2.A variant AHAS protein as defined in claim 1, wherein said herbicide isselected from the group consisting of imidazolinones, sulfonylureas,triazolopyrimidine sulfonamides, pyrimidyl-oxy-benzoic acids,sulfamoylureas, sulfonylcarboximides, and combinations thereof.
 3. Avariant AHAS protein as defined in claim 1, wherein said AHAS protein isderived from Arabidopsis thaliana.
 4. A variant AHAS protein as definedin claim 1, wherein said variant AHAS has more than about 20% of thecatalytic activity of wild type AHAS.
 5. A variant AHAS protein asdefined in claim 1, wherein said variant AHAS is at least 2-fold moreresistant to imidazolinone-based herbicides than to sulfonylurea-basedherbicides.